Production of 7-amino-3-methylcephem compounds

ABSTRACT

PROCESS FOR CONVERTING 7-ACYLAMIDO-3-METHYL-3-CEPHEM4-CARBOXYLIC ACID OR ITS ESTER TO THE CORRESPONDING 7-AMINO COMPOUND OR ITS ESTER BY BRINGING THE 7-ACYLAMIDO COMPOUND INTO CONTACT WITH, FOR EXAMPLE, A CULTURE BROTH OF A MICROORGANISM BELONGING TO THE GENUS ESCHERICHIA, BACILLUS, XANTHOMONAS, PSEUDOMONAS, PROTAMINOBACTER, MYCOPLANA OR AEROMANOAS. ALSO PROVIDED ARE THE NAMES OF SEVERAL OTHER MICROORGANISMS USEFUL IN THIS PROCESS.

United States Patent 3,749,641 PRODUCTION OF 7- MINO-3-METHYLCEPHEM COMPOUNDS Takeshi Takahashi and Kenji Kawahara, Osaka, and

Masao Isono, Hyogo, Japan, assignors to Takeda Chemical Industries, Ltd., Osaka, Japan No Drawing. Filed Dec. 20, 1971, Ser. No. 210,105 Claims priority, application Japan, Dec. 25, 1970, 45/129,648 Int. Cl. C12b 1/00 U.S. Cl. 195-29 11 Claims ABSTRACT OF THE DISCLOSURE Process for converting 7-acylamido-3-methyl-3-cephem- 4-carboxylic acid or its ester to the corresponding 7 -amino compound or its ester by bringing the 7-acylamido compound into contact with, for example, a culture broth of a microorganism belonging to the genus Escherichia, Bacillus, Xanthomonas, Pseudomonas, Protaminobacter, Mycoplana or Aeromonas. Also provided are the names of several other microorganisms useful in this process.

This invention relates to production of 7-amino-3- methylcephem compounds. More particularly, the present invention is concerned with a method for producing 7-amino-3-methyl-3-cephem-4-carboxylic acid of the Formula I or its ester from 7-acylamido-3-methyl-3-cephem-4-carboxylic acid or its ester by an enzymatic N-deacylation.

The desired product, 7-amino-3-n1ethyl-3-cephem-4- carboxylic acid of the formula:

or its ester at the 4-carboxyl, is known as an important intermediate for the syntheses of cephalosporins having a broad antimicrobial spectrum such as cephalexin. It has been known that the Compound I or its ester can be produced chemically by the hydrolysis of 7-phenoxyacetamido-3-methyl-3-cephem-4-carboxylic acid ester or by a reductive elimination of the acetoxy group from 7-aminocephalosporanic acid or its ester. The former process, however, involves difiiculties in specifically splitting the terminal acid amide linkage at the 7-position without affecting other parts of the molecule, and particularly when the Compound I is desired, the ester residue is required to be a special radical so as to be easily hydrolyzed. Furthermore, the yield of the desired compound varies with even a slight deviation of the reaction conditions. The latter process gives a still less yield than the former process. As far as the known processes are concerned, therefore, none has been industrially feasible.

The present inventors have extended the former process, but in a quite diiferent way by means of an enzyme system of microorganisms, and have succeeded in establishing an industrially feasible method according to which the 7-acylamido-3-methyl-3-cephem-4-carboxylic acid or its ester receives a cleavage of the acid amide linkage at the 7-position, and when the ester is used as the substrate, the ester linkage at the 4 position may further be cleaved to give the Compound I.

As a result of the research made on a variety of microorganisms, the present inventors have found that there exist those microorganisms which can produce the desired 7-amino-3-methyl-3-cephem-4-carboxylic acid or its ester from the 7-acylamido-3-methyl-3-cephem-4-carboxylic acid or its ester, and that these microorganisms are widely distributed, in nature or in culture collections, among fungi, yeasts and bacteria inclusive of actinomycetes.

It has also been found that said conversion is due to the capacity of the microorganisms that can hydrolyze solely the acid amide linkage in case of the substrate being the 4-carboxylic acid and that can hydrolyze either solely the acid amide linkage or both of the acid amide linkage and the carboxylic acid ester linkage in case of the substrate being the 4-carboxylic acid ester.

According to the present inventors survey, it has been confirmed that such microorganisms exist Widely in such genera as Aureobasidium, Anthracobia, Ascotricha, Acremonium, Actinomucor, Alternaria, Aegerita, Beauveria, Botryotinia, Byssachlamys, Botryosphaeria, Corynespora, Cunninghamella, Cylindrocarpon, Cylindrocladium, Colletotrichum, Cylindrocephalum, Cercospora, Ceratocystis, Cephalosporium, Cladosporium, Coniochaeta, Chaetomium, Chrysosporium, Cephaloascus, Diaporthe, Daedaleopsis, Dactylaria, Emericellopsis, Fusarium, Griphosphaeria, Gibberella, Glomerella, Isaria, Microascus, Neurospora, Pestalotiopsis, Pyrenophora, Pseudoplea, Papularia, Pleospora, Pellicularia, Rosellinia, Septoria, Sclerotium, Sartorya, Trichometasphaeria, Taphrino, Talaromyces, Verticillium, Nocardia, Streptomyces, Escherichia, Bacillus, Xanthomonas, Pseudomonas, Protaminobacter, Mycoplana, Aeromonas, Acetobacter, Rhodotorula, Cryptococcus, Nadsonia, Saccharomycodes and Saccharomyces. In view of the efiicacy of the transforming activity, i.e. of the reaction velocity, the preferable microorganisms are bacteria, particularly those belonging to the genus Escherichia, Bacillus, Xanthomonas, Pseudomonas, Prot aminobacter, Mycoplana or Aeromonas.

According to the method of this invention, 7-phenylacetamidoor 7-phenoxyacetamido-3-methyl-3-cephem-4- carboxylic acid or its ester is brought into contact with either of a culture broth of said microorganism or a processed matter thereof in an aqueous medium.

Thus, the starting compound used as the substrate is 7 phenylacetamido-3-methyl-3-cephem-4-carboxylic acid, 7-phenoxyacetamido-3-methyl-3-cephem-4-carboxylic acid or an ester thereof, and is produced, for example, by conversion from the corresponding 6 acylamidopenicillanic acid esters via their S-oxides as described in U.S. Pat. No. 3,275,626 and Belgian aPt. No. 747,118.

The starting compound may be put in use in the form of the free acids or the sodium, potassium, ammonium, amine or other salts. Examples of the esters as the starting compound are alkyl esters (e.g. methyl, ethyl, butyl or hexyl ester), substituted alkyl esters (e.g. methylthiomethyl, methylsulfenylmethyl, methylsulfonylmethyl, chloromethyl, bromomethyl or trichloroethyl ester; i.e. the substituent being alkylthio, alkylsulfenyl, alkylsulfonyl, halo and so on), aralkyl esters (e.g. benzyl or phenethyl ester) or other esters.

Said microorganism can be selected from the collected cultures available in public facilities for culture collection, or can also be isolated from soils, sewages, sea waters, flowers, fruits, air or other sources by per se conventional means. According to the present inventors experiences, said selection of the usable microorganism is readily effected, for example, by the following procedures:

PROCEDURE A Each of the candidate strains is aerobically cultivated in 20 m1. of an aqueous medium under the respective conditions as illustrated in Table 1 according to Whether the strains are bacteria, actinomycetes, fungi or yeasts.

TABLE 1 Microorganism Culture medium composition Cultivation Bacteria 1% meat extract, 1% peptone, 0.5% monosodium L-glutamate, 0.05% phenylacetic acid (or phe- At 28 0. to 37 C.

noxyacetie acid as the case may be), p 7.2. for 1 to 2 days.

Fungi or actinomycetcs 3% sucrose, 0.2% NaNO 0.1% KzHPoi, 0.05% KCl, 0.05% MgSOMH O, 0.0i% FeSO4-7HzO, At 28 C. for 4 days.

0.5% yeast extract, 0.5% peptone, 0.5% corn steep liquor, 0.05% phenoxyacetic acid (or phenylacetic acid as the case may be),

pH 6.0. Yeasts 5% sucrose, 0.025% KH2PO4, 0.1% CaNOs, 0.025% MgSO4-7HQO, 0.012% KCl, pH 6.0 At 28 C. for 2 days To the culture is added a solution of 20 mg. of a substrate (say methyl 7-phenylacetamidoor 7-phenoxyacetamido- 3-methyl-3-cephem-4-carboxylate) in 0.4 ml. of acetone, and the mixture is then shaken at 28 C. for 16 hours. A tiny portion (e.g. 0.01 ml.) of the resulting mixture is subjected to a thin-layer chromatography on silica gel and is developed with methanolzacetone (1:2 by volume) as long as 10 cm. The chromatogram is irradiated by ultraviolet rays to find the relevant spots. Under the specific conditions as above, the respective Rf-values of the substrate and the products can previously be determined as for example;

086 for methyl 7 phenylacetamidoor 7-phenoxyacetamido-3-methyl-3-cephem-4-carboxylate,

0.65 for methyl 7-amino-3-methyl-3-cephem-4-carboxylate,

0.42 for 7-amino-3-methyl-3-cephem-4-carboxylic acid.

Thus, in the above example, the reaction mixture which gave a spot or spots at Rf 0.65 and/or Rf 0.42 is selected, and is allowed to pass through a column of resin beads of styrene-divinylbenzene copolymers (e.g. commercially available as Amberlite XAD-Z, Rohm & Haas Co., U.S.A.) of ca. 10 mm. in diameter by 100 mm. in length. Fractions eluted by 40 ml. of water are collected and the total yield of the products is measured by means of ultraviolet absorption at the wavelength of 260 millimicrons. When the measurement shows that the yield is 10% or higher relative to the amount of the employed substrate, the candidate organism used to give the reaction mixture is judged to be usable for the method of the present invention.

PROCEDURE B (SIMPLIFIED PROCEDURE) In the same way a culture of each of the candidate strains is prepared. To 5 ml. of the culture broth is added a solution of 50 mg. of a substrate (e.g. 7-phenylacetamide-3-methyl-3-cephem-4-carboxylic acid) in 1 ml. of a 0.25 M phosphate buffer (pH 7.0). The mixture is then shaken at 28 C. for 16 hours. As to the resultant mixture the following three tests are applied:

(1) Whether the antibacterial activity against Bacillus subtilis PCI-2l9 of the resultant mixture has dropped to 80% or less of the activity of the same before the reaction, by appropriate modifications of the paper disc plate method of Higgins et al., Antibiotic and Chemotherapy, 3, 50-54 (1953) and Loo et al. Journal of Bacteriology, 50, 701-709 (1945);

(2) Whether the antibacterial activity of the resultant mixture will increase by the phenylacetylation with phenylacetyl chloride according to appropriate modification of Batechelors et al. method, Nature, 183, 257-258 (1959);

(3) Whether the absorbance of the resultant mixture at the wave length of 260 millimicrons has amounted at least 20% relative to that of the mixture before the reaction.

The candidate organisms which gave affirmative answers to all of the three tests can be used for the method of the present invention.

According to the practices by the present inventors both of Procedure A and Procedure B give substantially the same judgement on the utilizability of the same candidate organism.

Said contact of the substrate with the culture broth or its processed matter may be effected in such a manner that the organism is cultivated in a culture medium containing the substrate if the organism has tolerance against the substrate. However, it is more generally adopted to cultivate such microorganism in a suitable manner and then make use of the resulting cultured broth or its processed matter. Cultivation may be carried out under stirring with aeration, under shaking or stationarily, but in any way an aerobic cultivation is generally preferable. Culture media are prepared by selecting the components, solely or in combination, according to necessity, from meat extract, yeast extract, peptone, casein hydrolyzate, corn steep liquor, potato juice or any other conventionarily used natural matters; carbon compounds such as sugars, organic acids or normal paraffins; a variety of inorganic and organic nitrogen-containing compounds in a form of amino or nitrate; phosphates, magnesium salts, table salt or other metallic ions and various vitamins. The addition of phenoxyacetic acid or phenylacetic acid, which corresponds to the acyl group of the substrate to be employed, to the culture media at a concentration of 0.05 to 1% may result in enhancing the activity of the resulting culture broth of producing the desired product from the substrate. Suitable pH of the culture media and suitable cultivation temperature varies according to the kind of the microorganisms. But a desirable-pH usually lies in a range of pH 5 to 8 for fungi or yeasts and pH 6 to 9 for bacteria or actinomycetes. The temperature is usually selected from about 10 to about 40 C. The time at which said activity of the culture broth reaches the maximum varies according to the kind of the microorganisms employed, so the optimum time for cultivation is desirably determined as to each of the strains. The culture broth thus obtained or a processed matter thereof is utilized for the production of 7-amino-3-methyl-3-cephem-4-carboxylic acid or its ester. As the processed matter of the culture broth, anything which has been processed by suitable means for elevating or concentrating said transforming activity, can be used. When said activity mainly exists intracellularly, for example, 1) a cell suspension of separated cells in a buffer solution, (2) a cell-free extract of the cells obtained by per se conventional means (e.g. as described in Section I, particularly Articles 7 and 9, of Methods in Enzymology, Vol. I, edited by S. P. Colowick and N. 0. Kaplan, published by Academic Press Inc., New York in 1955) or (3) an enzyme solution purified or partially purified from said cell-free extract by per se known means (e.g. as in the subsequent Articles 10 to 13, supra), are suitably utilized. When said activity is mainly found to be extracellularly accumulated, (1) a supernatant solution obtained by the cells or mycelia from the cultured broth or (2) an enzyme solution purified or partially purified from said supernatant solution by applying the said known means for purification of enzymes may be exemplified.

The contact of the substrate with thus prepared cultured broth or its processed matter is conducted in water or any other aqueous medium. The pH of the reaction media is desirably adjusted to be between 4 and 9, more particularly between 6 and 8. Suitable reaction time is affected by concentrations of the substrates, the transforming activity of the culture broth or of its processed matter, reaction temperature and so forth, but is usually sufficient with 1 to 24 hours. The reaction temperature is selected between 10 and 50 C. and is desirably between 15 and 40 C. Concentration of the substrate is mainly determined in relation to the intensity of transforming activity for producing the desired compound, but usually selected from a range of 0.1 to 5% (weight/volume) in terms of the starting compound to the medium.

The 7-amino-3-methyl-3-cephem 4 carboxylic acid or its ester can be isolated and purified under mild conditions by combination of per se known means such as extraction with solvents or chromatography. It is particularly recommended to subject the filtrate of the reaction mixture to a column chromatography on Amberlite XAD2 and the elution with water to collect the fractions containing the desired product.

The method of this invention will further be explained in detail by way of the following examples. In the examples as well as in the foregoing description, the abbreviations mm, mL, mg, g, M and N" mean millimeter(s), milliliter(s), milligram(s), gram(s), molar concentration and normal, respectively; the percentages in respect of the culture medium compositions are on a weight per volume basis, i.e. grams per deciliter, and those in other respects are on a weight basis unless otherwise noted. Part by weight bears the same relationship to part by volume as gram does to milliliter. In the tables, means that the product was not detectable. The IFO numbers and ATCC number attached to the respective species names are the accession numbers in Institute for Fermentation, Osaka, Japan and in American Type Culture Collection, Maryland, U.S.A. and all strains had been available prior to this invention as listed in lists of cultures published by the respective culture collections, except for the following which are not listed in the publications but have deposited also at the Americal Type Culture Collection under the respective accession numbers:

Escherichia coli IFO3210: ATCC-21758 Escherichia coli IFO -3467: ATCC-21753 Xanthomonas oryzae IFO-3312: ATCC-21754 Protamin-obacter alboflavus TI O-13221: ATCC-21755 Mycoplana sp. IFO-l3240: ATCC-21756 Aeromonas hydrophila IFO-12634: ATCC-21757 As to the Mycoplana sp. IFO-13240, the species has not yet been fixed and thus the characteristics of the strain are described as follows:

Irregular rods of 0.6 micron by 2 to -6 microns, sometimes curved, and branched when very young. Motile by means of polar flagella. Gram-negative: Aerobic. Agar colonies: Circular, light gray, convex smooth,

entire. Agar slant: Filiform, light gray, smooth. Broth: Turbid. Gelatin stab: No liquefaction. Thin surface growth. Casein, starch and cellulose were not hydrolyzed. Nitrites not produced from nitrates. Hydrogen sulfide not produced. Indole not produced. Methyl Red test: Negative. Catalase: Positive. Litmus milk: Unchanged. Potato: No growth. No growth or scant, if any, in carbohydrate media. Aromatic compounds such as phenylglycine were utilized. Isolated from soil.

EXAMPLE 1 A 2-day inocul-um (5 parts by volume) of Escherichia coli IFO-3210 was inoculated in 200 parts by volume of a culture medium of pH 7.2, comprising 1.0% of meat extract, 1.0% of peptone, 0.5% of sodium glutamate, 0.5 of sodium chloride and 0.05% of phenylacetic acid. The culture medium was incubated at 37 C. under shaking for 48 hours, followed by the addition of a solution of 0.8 part by weight of methyl 7-phenylacetamido- 3 methyl 3 cephem 4 carboxylate in 8 parts by volume of acetone together with 50 parts by volume of a 0.25 M phosphate buffer solution (pH 7.0). The whole mixture was then shaken at 28 C. for 16 hours, whereupon the methyl 7 phenylacetamido 3 methyl 3- cephem 4 carboxylate disappeared completely in the mixture and 0.4 part by weight of 7-amino-3-methyl-3- cephem-4-carboxylic acid was produced therein.

The reaction mixture was filtered to remove the cells, and the filtrate was subjected to a column chromatography on Amberlite XAD-Z (vide supra) with the use of distilled water as the eluant, to give 0.35 part by weight of 7-amino 3 methyl 3 cephem 4 carboxylic acid, melting at 240 to 242 C. with decomposition.

EXAMPLE 2 In the same manner as in Example 1, the respective 2-day cultures of the bacterial strains listed in Table 2 were prepared. To 200 ml. of the respective culture broths were added a solution of 800 mg. of methyl 7-phenylacetamido 3 methyl 3 cephem 4 carboxylate (Substrate 1) in 8 ml. of acetone and 50 ml. of a 0.25 M phosphate buffer solution (pH 7.0). Alternatively, to another 200 ml. of each of the culture broths was added a solution of 800 mg. of 7-phenylacetamido 3 methyl- 3-cephem 4 carboxylic acid (Substrate 2) in 50 ml. of a 0.25 M phosphate buffer (pH 7.0). The mixtures were further shaken at 28 C. for 16 hours to allow the reaction to take place. Products were separated from the reaction mixture and purified by column chromatography. The yields were determined on the absorptions at 260 millimicrons, as shown in Table 2, wherein:

Product A: Methyl 7-amino-3-methyl-3-cephem-4-carboxylate Product B: 7-Amino-3-methyl-3-cephem 4 carboxylic acid A loopful of each of the bacterial strains listed in Table 3 was inoculated in 5 ml. of an aqueous medium (pH 7.0) comprising 0.8% of Bacto-Nutrient Broth, dehydrated (commercially available, distributed by Difco Laboratories, USA.) and 0.3% of phenylacetic acid, and was then cultivated under shaking at 28 C. for 24 hours. To the culture broth was added a solution of 30 mg. of either 7-phenylacetamido 3 methyl 3 cephem-4- carboxylic acid (Substrate 1) or 7-phenoxyacetamido-3- methyl 3 -cephem 4 carboxylic acid (Substrate 2) in 1 ml. of a 0.25 M phosphate buffer (pH 7.0). The shaking was further continued at 28 C. for 4 hours as to Substrate 1 and for 24 hours as to Substrate 2, Whereupon the reaction mixture came to show no antimicrobial activity. This means that the substrates were completely transformed. The yield of 7-amino 3 methyl-3-cephem- 4-carboxylic acid was determined by means of the restored antimicrobial activity after phenylacetylation with phenylacetyl chloride. The results are shown in Table 3.

7 EXAMPLE 4 A loopful of Escherichia coli IFO-32l0 was inoculated in 50 parts by volume of an aqueous culture medium (pH 7.0) comprising 1% of peptone, 1% of meat extract, 0.5% of NaCl and 0.3% of phenylacetic acid, and was cultivated under shaking at 28 C. for 24 hours to prepare a seed culture. The seed was then inoculated in 500 parts by volume of an aqueous medium of the same composition as above-used, and the whole mixture was shaken at 28 C. for 24 hours. The culture broth was filtered to collect the cells, which were then washed with water and suspended in 50 parts by volume of distilled water.

To the cell suspension was added 50 parts by volume of an aqueous solution of 2 parts by weight of 7-phenylacetamido 3 methyl-3-cephem-4-carboxylic acid. The mixture was stirred at 37 C. for 3 hours under adjustment of pH to 7.0 by intermittent addition of 0.1 N NaOH. At each 30 minute interval, a small portion of the reaction mixture was taken out and was measured its absorbance at 260 millimicrons. The same portion was then subjected to thin-layer chromatography on silica gel with a mixture of ethyl acetatezacetic acidzwater (50:5:4 by volume) as the developer. From the results, it turned that the substrate (Rf 0.50) disappeared in the 3-hour reaction completely to give 7-amino-3-methyl-3-cephem- 4-carboxylic acid (Rf 0.05). The absorbance at 260 millimicrons of the reaction mixture was not substantially changed during the reaction.

The reaction mixture was filtered to remove the cells, and the filtrate was combined with the washing of the cells. The combined solution was chromatographed on Amberlite XAD-Z, and the column was eluted with distilled water. Lyophilization of the eluates gave 1.0 part by weight of pure 7-amino-3-methyl-3-cephem-4-carboxyli: acid as powder showing good accord with an authentic sample with respect to melting point, specific rotation, infrared spectrum, nuclear magnetic resonance spectrum and ultraviolet spectrum.

EXAMPLE 5 A loopful of the respective 7-day slant cultures of the microorganisms listed below was inoculated into ml. each of a culture medium of pH 6, comprising 3% of sucrose, 0.2% of sodium nitrate, 0.1% of dipotassium hydrogenphosphate, 0.05% of potassium chloride, 0.05% of magnesium sulfate, 0.001% of ferrous sulfate, 0.5% of yeast extract, 0.5% of peptone, 0.5% of corn steep liquor and 0.05% of phenoxyacetic acid. The culture media were incubated under shaking at 28 C. for 4 days, followed by the addition of a solution of 100 mg. of methyl 7-phenoxyacetamido-3-methyl-3-cephem 4 carboxylate in 1 ml. of acetone together with 7 ml. of a 0.25 M phosphate buffer solution of pH 7.0. The reaction was conducted at 28 C. under shaking for 16 hours. The product or products by each of the strains are shown in Table 4, wherein the figures demonstrate the respective turnovers to the desired 7-amino-3-methyl-3-cephem-4- carboxylic acid (Product I) and/or methyl 7-amino-3- methyl-3-cephem-4-carboxylate (Product II).

TABLE 4 Turnover (percent) TABLE 4---Continued Turnover (percent) Product Product I II Microorganism Botryosphaerz'a rz'bis chromogena IFO-4837 17 Oun'ninghamella echinulata IFO-4445 50 C z'chum IFO-67 13 Cylindrocephalum aureum IFO-6807 45 Carcospora IFO-6711 Ceratocystis piceae IFO-7644 Oer IFO-86fi Cephalosporium mz/cophz'lum IFO-6615 Ohaetomium elatu'm IFO-6554 Chaetomiu'm anahelicinum IFO-8389 Ohrysosporium verrucosum IFO8279 Cephaloaacus fragrans IFO-7785 Corynespora smithii 11 0-8162 Cylindrocladium penicilloidea U O-6784.. Cylindrocarpon willkom'rm'i IFO5995.. Cladosporium cladosporioides IFO6371 Coniochaeta teiraspora IFO8526.. Dactylaria mycophila IFO-6785-.. Diaporthe phaseolorum IFO-6707- Daedaleopsts styracina IFO4910.- Fuaarium roseum IFO-8503 Fusarz'um solani UFO-8509 Griphosphaen'a m'valis IFO Gibberella zcae [FD-7100 Glomerella cingulata IFO Isarz'a kogane IFO- fl Microascus desmosporus IFO-6761.. Neurospom sitophila IFO-4596 Pestalotiopsis funerea IFO5427 Pleospora herbarum IFO-6125 Pyrenophora graminea IFO-7507- Pseudoplea trifoliz IFO-6681 Papularia sphaerosperma IFO-657 Pellz'cularz'a filamentosa IFO-6523. Rosellim'a necatriz' IFO-6323- Septoria glycines IFO-7346 Sclerotium delphinii IFO7337. Sartorya fumigata IFO-5866 irichometasphaeria turcica IFO-6358 Taphrina cerasi IFO-0675 Talaromycea Zuteus IFO-6896--. Verticz'llium theobromae IFO-666 Nocardia mezicana IFO-3927-..

Streptomyces Zavendulae IFO-3l45 EXAMPLE 6 A 4-day seed culture (30 parts by volume) of Griphosplzaeria nivalis IFO-7436 was inoculated into 1,000 parts by volume of a culture medium of pH 6, which comprised 3% of sucrose, 0.2% of sodium nitrate, 0.1% of dipotassium hydrogenphosphate, 0.05 of potassium chloride, 0.05 of magnesium sulfate, 0.001% of ferrous sulfate and 0.05% of phenoxyacetic acid. The culture medium was incubated under shaking at 28 C. for 4 days, followed by the addition of a solution of 3.7 parts by weight of methyl 7-phenoxyacetamido-3-methyl-3-cephem- 4-carboxylate in 20 parts by volume of acetone. The mixture was further shaked at 28 C. for 16 hours.

A part of the reaction mixture was subjected to thin layer chromatography with the use of methanohacetone 1:1) as the solvent, to find that the substrate methyl 7- phenoxyacetamido-3-methyl-3-cephem-4-carboxylate (Rf 0.95) had disappeared and that there had been instead produced 7 amino-3-methyl-3-cephem-4-carboxylic acid with an almost quantitative yield.

The reaction mixture was filtered to remove the mycelia, and the filtrate was concentrated and purified by column chromatography to give 1.9 part by weight of the product, melting at 240 to 242 C. with decomposition.

EXAMPLE 7 A loopful of a 4-day agar slant culture of each of the yeast strains listed in Table 5 was inoculated into 30 ml. of a culture medium of pH 6, consisting of 5% of sucrose, 0.025% of potassium dihydrogenphosphate, 0.1% of calcium nitrate, 0.025% of magnesium sulfate, 0.012% of potassium chloride and tap water. Cultivation was conducted at 28 C. under shaking for 48 hours. To the culture medium were added a solution of mg. of methyl 7-phenoxyacetamido-3-methyl-3-cephem 4 carboxylate in 1 ml. of acetone and 7 ml. of a 0.25 M phosphate buffer solution (pH 7.0), and the shaking was further continued at 28 C. for 16 hours to allow the reaction to take place. The yields of the resulting product, 7-amino-3-methyl-3- cephem-4-carboxylic acid (Product I) or its methyl ester (Product II), by the respective strains are shown in the following table.

EXAMPLE 8 To 30 ml. of a 4-day culture broth of each of the strains listed in Table 6, prepared in the same manner as in Example 5, there were added 7 ml. of a 0.25 M phosphate buffer solution (pH 7.0) and a solution of 100 mg. of benzyl 7 phenoxyacetamido-3-rnethyl-3-cephem-4-carboxylate in 1 ml. of acetone. The mixture was shaken at 28 C. for 16 hours to allow the reaction to take place. The yields of the resulting 7-amino-3-methyl-3cephem-4- carboxylic acid (Product I) and its benzyl ester (Product II), in terms of respective turnovers are shown in Table 6.

EXAMPLE 9 To 30 ml. of a 4-day culture broth of each of the strains listed in Table 7, each prepared in the same manner as in Example 5, there were added 7 ml. of a 0.25 M phosphate buffer solution (pH 7.0) and a solution of 100 mg. of methylthiomethyl 7 phenoxyacetamido 3 methyl-3- cephem-4-carboxylate.

The mixture was shaken at 28 C. for 16 hours to allow the reaction to take place. The yields of the resulting 7- amino 3 methyl-3-cephem-4-carboxylic acid (Product I) and/ or its methylthiomethyl ester (Product II) in turnovers are shown in Table 7.

'EXAMPLE 10 Escherichia coli IFO-3210 was cultivated in the same manner as in Example 1 for 24 hours. The cultured broth in an amount of 200 parts by volume was centrifuged to collect cells. The cells were suspended in 20 parts by volume of a 0.05 M phosphate buffer solution (pH 7.0) and then subjected to a sonication of 10 kilocycles per second at C. for 20 minutes. The resultant sonicates were centrifuged to obtain 17 parts by volume of a cellfree supernatant.

To the supernatant was added a solution of 0. 4 part by weight of 7-phenylacetamido 3 methyl-3-cephem-4- carboxylic acid in 23 parts by volume of a 0.05 M phosphate buifer (pH 6.0), and the mixture was shaken at 37 C. for 5 hours, whereupon 0.19 part by weight of 7-amino- 3-methyl-3-cephem-4-carboxylic acid was produced.

We claim:

1. A method for producing 7 amino 3 methyl-3 acid or its ester, the acyl portion of the 7-acylamido group thereof being phenylacetyl or phenoxyacetyl, into contact with a culture broth of a microorganism selected from the group consisting of Aureobasidium, Anthracobia, Ascotricha, Acremonium, Actinomucor, Alternaria, Aegerita, Beauveria, Botryotinia, Byssochlamys, Botryosphaeria, Corynespora, Cunninghamella, Cylindrocarpon, Cylindrocladium, Colletotrichum, Cylindrocephalum, Cercospora, Ceratocystis, Cephalosporium, Cladosporium, Coniochaeta, Chaetomium, Chrysosporium, Cephaloascus, Diaporthe, Daedaleopsis, Dactylaria, Fusarium, Griphosphaeria, Gibberella, Glomerella, Isaria, Microascus, Neurospora, Pestalotiopsis, Pyrenophora, Pseudoplea, Papularia, Pleospora, Pellicularia, Rosellinia, Septoria, Sclerotium, Sartorya, Trichometasphaeria, Taphrina, Talaromyces, Verticillium, Nocardia, Escherichia, Bacillus, Xanthomonas, P'seudomonas, Protaminobacter, Mycoplana, Aeromonas, Nadsonia, Saccharomycodes, Arthrinium phaeospermum, Aspergillus parasiticus and Aspergillus brevipes or the enzyme-containing matter thereof, in an aqueous medium, said microorganism being selected from those capable of producing 7-amino-3-methyl-3-cephem- 4 carboxylic acid or its ester from the 7 acylamido-3- methyl-3-cephem-4-carboxylic acid or its ester by the hydrolysis of only the acid amide linkage in the case of the 4-carboxylic acid, or by the hydrolysis either of the acid amide linkage alone or of both the acid amide linkage and the carboxylic acid ester linkage in the case of the 4- carboxylic acid ester.

2. The method according to claim 1, wherein said contact is eifected at a pH between 4 and 9 at a temperature between 10 C. and 50 C.

3. The method according to claim 1, wherein said microorganism is a bacterium belonging to the genus Escherichia, Bacillus, Xanthomonas, Pseudomonas, Protaminobacter, Mycoplana or Aeromonas.

4. The method according to claim 1, wherein the substrate is brought into contact with a culture broth of said microorganism.

5. The method according to claim 1, wherein the substrate is brought into contact with a cell suspension of said microorganism.

6. The method according to claim 3, wherein the bacterium is Escherichia coli EEO-3210.

7. The method according to claim 3, wherein the bacterium is Escherichia coli 11 0-3467.

8. The method according to claim 3, wherein the bacterium is Xanthomonas malvacearum IF O-3383.

9. The method according to claim 3, wherein the bacterium is Bacillus pumz'lus IFO12093.

10. The method according to claim 3, wherein the bacterium is Bacillus licheniformis JED-12199.

11. The method according to claim 3, wherein the bacterium is Bacillus sp. ATCC-14552.

References Cited UNITED STATES PATENTS 9/ 1964 Kleinschmidt et a1. 19536 P 9/1970 Cole et a]. 19536 P US. Cl. X.R. 195-36 P, R 

